Development of serotyping reagents for the periodontopathogenic bacterium, Bacteroides (Porphyromonas) gingivalis.

Principal Investigator

NAKAMURA Ryo The University of Tokushima, School of Dentistry, Professor, 歯学部, 教授 (30034169)

Co-Investigator(Kenkyū-buntansha)

MAN-YOSHI Tomie The University of Tokushima, University Dental Hospital, Research Associate, 歯学部附属病院, 助手 (20200644)
木戸 玲子 徳島大学, 歯学部附属病院, 講師 (30181605)
NAGATA Atsushi The University of Tokushima, University Dental Hospital, Assistant Professor, 歯学部附属病院, 講師 (70228021)
SATO Makoto The University of Tokushima, School of Dentistry, Associate Professor, 歯学部, 助教授 (10126229)
ICHIMIYA Seiko The University of Tokushima, University Dental Hospital, Research Associate, 歯学部附属病院, 助手 (30223845)
HINODE Daisuke The University of Tokushima, School of Dentistry, Research Associate, 歯学部, 助手 (70189801)

Bacteroides (Porphyrosonas) gingivilis has been considered one of the most virulent bacteria in relation to adult periodontitis. This bacterium has been serologically characterized to have at least four types with different biological activities such as collagenolytic and proteolytic enzymes and antibiograss. The purpose of this study is to develop the serotyping reagents by producing monoclonal antibodies (MAbs) for specific antigens of these four serotypes of lacteroides gingivalis.BALB/c mice were intraperitoneally immunized with serotype representative strains (381 for serotype I, JH4 for serotype II, 22KNB-12 for serotype IIII nd 16NH2-1 for serotype IV) in Freund incomplete adjuvant. Spleen cells from immunized mouse were fused with P3X6-Ag8. U1 (P3U1) myelona cells usins 45% PEG, 10% DMSO in IMDM. After selecting hybrid cells by HAT selection, clones producing NAbs that reacted only with a strain used for the immunization were screened by ELISA.Among MAbs produced by hybridomas
… Morewhich developed a positive reaction in ELISA, 8 MAbs reacted with the strain 381, 22 with JH4, 3 with 22KN6-12 and 8 with 16NH2-1, respevtively. Five out of 8 MAbs, which reacted with a strain 381, developed a positive reaction also with other serotype I strains including reference and freshly isolated ones, without cross-reacting with other serotype group strains. Similarly, 4 out of 22 MAbs against JH4 and 1 out of 8 MAbs against 16NH2-1 reacted with other strains belonging to their corresponding serotype groups. Some of these MAbs were heat-labile and others were heat-stable, suggesting that antigenic determinants consist of either peptide or polysaccharide. SDS-PAGE patterns of bacterial extract from 4 serotype groups were almost similar, but when the MAb was reacted with heat-treated (heat-stable) antigens by a Western blotting procedure, MAb asainst strains from serotype II and IV reacted with an antigen of 50 and 100 KDa from the same serotype strain, respectively, suggesting that these bands could be an serotype specific antigens. MAbs obtained in this study might be useful in classifying infectious features of the patients with adult periodontitis.さらにWestern blot法でみてみると、易熱性のものは、反応するバンドが検出できなくなることが多いが、熱耐性のものは血清型IIのMAb X2H3が50KDa、血清型IVのMAb P2E6が100KDaのバンドとのみ反応を示した。これらのバンドはSDSーPAGEで4つの血清型菌に共通にみられるバンドであるが他の型の菌に対して反応しないことから特異抗原あるいはその一部であることが示唆された。 Less